Drug screening on digital microfluidics for cancer precision medicine.

Drug screening based on in-vitro primary tumor cell culture has demonstrated potential in personalized cancer diagnosis. However, the limited number of tumor cells, especially from patients with early stage cancer, has hindered the widespread application of this technique. Hence, we developed a digital microfluidic system for drug screening using primary tumor cells and established a working protocol for precision medicine. Smart control logic was developed to increase the throughput of the system and decrease its footprint to parallelly screen three drugs on a 4 × 4 cm2 chip in a device measuring 23 × 16 × 3.5 cm3. We validated this method in an MDA-MB-231 breast cancer xenograft mouse model and liver cancer specimens from patients, demonstrating tumor suppression in mice/patients treated with drugs that were screened to be effective on individual primary tumor cells. Mice treated with drugs screened on-chip as ineffective exhibited similar results to those in the control groups. The effective drug identified through on-chip screening demonstrated consistency with the absence of mutations in their related genes determined via exome sequencing of individual tumors, further validating this protocol. Therefore, this technique and system may promote advances in precision medicine for cancer treatment and, eventually, for any disease.

Definition of a new blood cell count score for early survival prediction for non-small cell lung cancer patients treated with atezolizumab: Integrated analysis of four multicenter clinical trials.

Importance: Blood cell count test (BCT) is a robust method that provides direct quantification of various types of immune cells to reveal the immune landscape to predict atezolizumab treatment outcomes for clinicians to decide the next phase of treatment. Objective: This study aims to define a new BCTscore model to predict atezolizumab treatment benefits in non-small lung cell cancer (NSCLC) patients. Design Setting and Participants: This study analyzed four international, multicenter clinical trials (OAK, BIRCH, POPLAR, and FIR trials) to conduct post-hoc analyses of NSCLC patients undergoing atezolizumab (anti-PD-L1) single-agent treatment (n = 1,479) or docetaxel single-agent treatment (n = 707). BCT was conducted at three time points: pre-treatment (T1), the first day of treatment cycle 3 (T2), and first day of treatment cycle 5 (T3). Univariate and multivariate Cox regression analyses were conducted to identify early BCT biomarkers to predict atezolizumab treatment outcomes in NSCLC patients. Main Outcomes and Measures: Overall survival (OS) was used as the primary end point, whereas progression-free survival (PFS) according to Response Evaluation Criteria in Solid Tumors (RECIST), clinical benefit (CB), and objective response rate (ORR) were used as secondary end points. Results: The BCT biomarkers of neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) at time point T3 and neutrophil-to-monocyte ratio (NMR) at time point T2 with absolute cutoff values of NLR_T3 = 5, PLR_T3 = 180, and NMR_T2 = 6 were identified as strong predictive biomarkers for atezolizumab (Ate)-treated NSCLC patients in comparison with docetaxel (Dtx)-treated patients regarding OS (BCTscore low risk: HR Ate vs. Dtx = 1.54 (95% CI: 1.04-2.27), P = 0.031; high risk: HR Ate vs. Dtx = 0.84 (95% CI: 0.62-1.12), P = 0.235). The identified BCTscore model showed better OS AUC in the OAK (AUC12month = 0.696), BIRCH (AUC12month = 0.672) and POPLAR+FIR studies (AUC12month = 0.727) than that of each of the three single BCT biomarkers. Conclusion and Relevance: The BCTscore model is a valid predictive and prognostic biomarker for early survival prediction in atezolizumab-treated NSCLC patients.

Elucidation of the Application of Blood Test Biomarkers to Predict Immune-Related Adverse Events in Atezolizumab-Treated NSCLC Patients Using Machine Learning Methods.

Background: Development of severe immune-related adverse events (irAEs) is a major predicament to stop treatment with immune checkpoint inhibitors, even though tumor progression is suppressed. However, no effective early phase biomarker has been established to predict irAE until now. Method: This study retrospectively used the data of four international, multi-center clinical trials to investigate the application of blood test biomarkers to predict irAEs in atezolizumab-treated advanced non-small cell lung cancer (NSCLC) patients. Seven machine learning methods were exploited to dissect the importance score of 21 blood test biomarkers after 1,000 simulations by the training cohort consisting of 80%, 70%, and 60% of the combined cohort with 1,320 eligible patients. Results: XGBoost and LASSO exhibited the best performance in this study with relatively higher consistency between the training and test cohorts. The best area under the curve (AUC) was obtained by a 10-biomarker panel using the XGBoost method for the 8:2 training:test cohort ratio (training cohort AUC = 0.692, test cohort AUC = 0.681). This panel could be further narrowed down to a three-biomarker panel consisting of C-reactive protein (CRP), platelet-to-lymphocyte ratio (PLR), and thyroid-stimulating hormone (TSH) with a small median AUC difference using the XGBoost method [for the 8:2 training:test cohort ratio, training cohort AUC difference = -0.035 (p < 0.0001), and test cohort AUC difference = 0.001 (p=0.965)]. Conclusion: Blood test biomarkers currently do not have sufficient predictive power to predict irAE development in atezolizumab-treated advanced NSCLC patients. Nevertheless, biomarkers related to adaptive immunity and liver or thyroid dysfunction warrant further investigation.

Assessment of chimeric antigen receptor T cytotoxicity by droplet microfluidics in vitro.

Chimeric antigen receptor T (CAR-T) cells are cytotoxic T cells engineered to specifically kill cancer cells expressing specific target receptor(s). Prior CAR-T efficacy tests include CAR expression analysis by qPCR or ELISA, in vitro measurement of interferon-γ (IFNγ) or interleukin-2 (IL-2), and xenograft models. However, the in vitro measurements did not reflect CAR-T cytotoxicity, whereas xenograft models are low throughput and costly. Here, we presented a robust in vitro droplet microfluidic assay for CAR-T cytotoxicity assessment. This method not only enabled assessment of CAR-T cytotoxic activity under different fluid viscosity conditions, but also facilitated measurement of CAR-T expansion and dissection of mechanism of action via phenotype analysis in vitro. Furthermore, our data suggested that label-free cytotoxicity analysis is feasible by acquiring data before and after treatment. Hence, this study presented a novel in vitro method for assessment of cellular cytotoxicity that could potentially be applied to any cytotoxicity experiment with varying solvent composition.

Molecular landscape and subtype-specific therapeutic response of nasopharyngeal carcinoma revealed by integrative pharmacogenomics.

Nasopharyngeal carcinoma (NPC) is a malignant head and neck cancer type with high morbidity in Southeast Asia, however the pathogenic mechanism of this disease is poorly understood. Using integrative pharmacogenomics, we find that NPC subtypes maintain distinct molecular features, drug responsiveness, and graded radiation sensitivity. The epithelial carcinoma (EC) subtype is characterized by activations of microtubule polymerization and defective mitotic spindle checkpoint related genes, whereas sarcomatoid carcinoma (SC) and mixed sarcomatoid-epithelial carcinoma (MSEC) subtypes exhibit enriched epithelial-mesenchymal transition (EMT) and invasion promoting genes, which are well correlated with their morphological features. Furthermore, patient-derived organoid (PDO)-based drug test identifies potential subtype-specific treatment regimens, in that SC and MSEC subtypes are sensitive to microtubule inhibitors, whereas EC subtype is more responsive to EGFR inhibitors, which is synergistically enhanced by combining with radiotherapy. Through combinational chemoradiotherapy (CRT) screening, effective CRT regimens are also suggested for patients showing less sensitivity to radiation. Altogether, our study provides an example of applying integrative pharmacogenomics to establish a personalized precision oncology for NPC subtype-guided therapies.

Cisplatin prevents breast cancer metastasis through blocking early EMT and retards cancer growth together with paclitaxel.

Cancer growth is usually accompanied by metastasis which kills most cancer patients. Here we aim to study the effect of cisplatin at different doses on breast cancer growth and metastasis. Methods: We used cisplatin to treat breast cancer cells, then detected the migration of cells and the changes of epithelial-mesenchymal transition (EMT) markers by migration assay, Western blot, and immunofluorescent staining. Next, we analyzed the changes of RNA expression of genes by RNA-seq and confirmed the binding of activating transcription factor 3 (ATF3) to cytoskeleton related genes by ChIP-seq. Thereafter, we combined cisplatin and paclitaxel in a neoadjuvant setting to treat xenograft mouse models. Furthermore, we analyzed the association of disease prognosis with cytoskeletal genes and ATF3 by clinical data analysis. Results: When administered at a higher dose (6 mg/kg), cisplatin inhibits both cancer growth and metastasis, yet with strong side effects, whereas a lower dose (2 mg/kg) cisplatin blocks cancer metastasis without obvious killing effects. Cisplatin inhibits cancer metastasis through blocking early steps of EMT. It antagonizes transforming growth factor beta (TGFß) signaling through suppressing transcription of many genes involved in cytoskeleton reorganization and filopodia formation which occur early in EMT and are responsible for cancer metastasis. Mechanistically, TGFß and fibronectin-1 (FN1) constitute a positive reciprocal regulation loop that is critical for activating TGFß/SMAD3 signaling, which is repressed by cisplatin induced expression of ATF3. Furthermore, neoadjuvant administration of cisplatin at 2 mg/kg in conjunction with paclitaxel inhibits cancer growth and blocks metastasis without causing obvious side effects by inhibiting colonization of cancer cells in the target organs. Conclusion: Thus, cisplatin prevents breast cancer metastasis through blocking early EMT, and the combination of cisplatin and paclitaxel represents a promising therapy for killing breast cancer and blocking tumor metastasis.

Targeting NF-κB Signaling for Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematologic malignancy in the world. Even though survival rates have significantly risen over the past years, MM remains incurable, and is also far from reaching the point of being managed as a chronic disease. This paper reviews the evolution of MM therapies, focusing on anti-MM drugs that target the molecular mechanisms of nuclear factor kappa B (NF-κB) signaling. We also provide our perspectives on contemporary research findings and insights for future drug development.

Genome-wide piggyBac transposon-based mutagenesis and quantitative insertion-site analysis in haploid Candida species.

Invasive fungal infections caused by Candida species are life threatening with high mortality, posing a severe public health threat. New technologies for rapid, genome-wide identification of virulence genes and therapeutic targets are urgently needed. Our recent engineering of a piggyBac (PB) transposon-mediated mutagenesis system in haploid Candida albicans provides a powerful discovery tool, which we anticipate should be adaptable to other haploid Candida species. In this protocol, we use haploid C. albicans as an example to present an improved version of the mutagenesis system and provide a detailed description of the protocol for constructing high-quality mutant libraries. We also describe a method for quantitative PB insertion site sequencing, PBISeq. The PBISeq library preparation procedure exploits tagmentation to quickly and efficiently construct sequencing libraries. Finally, we present a pipeline to analyze PB insertion sites in a de novo assembled genome of our engineered haploid C. albicans strain. The entire protocol takes ~7 d from transposition induction to having a final library ready for sequencing. This protocol is highly efficient and less labor intensive than alternative approaches and significantly accelerates genetic studies of Candida.

A digital microfluidic system with 3D microstructures for single-cell culture.

Despite the precise controllability of droplet samples in digital microfluidic (DMF) systems, their capability in isolating single cells for long-time culture is still limited: typically, only a few cells can be captured on an electrode. Although fabricating small-sized hydrophilic micropatches on an electrode aids single-cell capture, the actuation voltage for droplet transportation has to be significantly raised, resulting in a shorter lifetime for the DMF chip and a larger risk of damaging the cells. In this work, a DMF system with 3D microstructures engineered on-chip is proposed to form semi-closed micro-wells for efficient single-cell isolation and long-time culture. Our optimum results showed that approximately 20% of the micro-wells over a 30 × 30 array were occupied by isolated single cells. In addition, low-evaporation-temperature oil and surfactant aided the system in achieving a low droplet actuation voltage of 36V, which was 4 times lower than the typical 150 V, minimizing the potential damage to the cells in the droplets and to the DMF chip. To exemplify the technological advances, drug sensitivity tests were run in our DMF system to investigate the cell response of breast cancer cells (MDA-MB-231) and breast normal cells (MCF-10A) to a widely used chemotherapeutic drug, Cisplatin (Cis). The results on-chip were consistent with those screened in conventional 96-well plates. This novel, simple and robust single-cell trapping method has great potential in biological research at the single cell level.

A Novel and Robust Single-cell Trapping Method on Digital Microfluidics.

Due to cell heterogeneity, the differences among individual cells are averaged out in bulk analysis methods, especially in the analysis of primary tumor biopsy samples from patients. To deeply understand the cell-to-cell variation in a primary tumor, single-cell culture and analysis with limited amount of cells are in high demand. Microfluidics has been an optimum platform to address the issue given its small reaction volume requirements. Digital microfluidics, which utilizes an electric signal to manipulate individual droplets has shown promise in cell-culture with easy controls. In this work, we realize single cell trapping on digital microfluidic platform by fabricating 3D microstructures on-chip to form semi-closed micro-wells. With this design, 20% of 30 x 30 array can be occupied by isolated single cells. We also use a low evaporation silicon oil and a fluorinated surfactant to lower the droplet actuation voltage and prevent the drop from evaporation, while allowing cell respiration during the long term of culture (24 h). The main steps for single cell trapping on digital microfluidics, as illustrated in this protocol, include 3D microstructures design, 3D microstructures construction on chip and oil film with surfactant for single cell trapping on chip.

Author Correction: Drug screening of cancer cell lines and human primary tumors using droplet microfluidics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Candida albicans gains azole resistance by altering sphingolipid composition.

In the original version of this Article, Haoping Liu, who conceptualized, designed and supervised the project and acquired funding, was inadvertently omitted from the author list. Furthermore, the affiliation of Jiaxin Gao and Haoping Liu with 'Department of Biological Chemistry, University of California, Irvine, CA 92697, USA' was omitted. Finally, funding from NIH grant GM117111, and contributions from Dr. Li-lin Du of NIBS for providing pPB[ura4] and pDUAL-PBase and Allan Bradley of Sanger for hyPBase, were not acknowledged. These errors have now been corrected in both the PDF and HTML versions of the Article.

Candida albicans gains azole resistance by altering sphingolipid composition.

Fungal infections by drug-resistant Candida albicans pose a global public health threat. However, the pathogen's diploid genome greatly hinders genome-wide investigations of resistance mechanisms. Here, we develop an efficient piggyBac transposon-mediated mutagenesis system using stable haploid C. albicans to conduct genome-wide genetic screens. We find that null mutants in either gene FEN1 or FEN12 (encoding enzymes for the synthesis of very-long-chain fatty acids as precursors of sphingolipids) exhibit resistance to fluconazole, a first-line antifungal drug. Mass-spectrometry analyses demonstrate changes in cellular sphingolipid composition in both mutants, including substantially increased levels of several mannosylinositolphosphoceramides with shorter fatty-acid chains. Treatment with fluconazole induces similar changes in wild-type cells, suggesting a natural response mechanism. Furthermore, the resistance relies on a robust upregulation of sphingolipid biosynthesis genes. Our results shed light into the mechanisms underlying azole resistance, and the new transposon-mediated mutagenesis system should facilitate future genome-wide studies of C. albicans.

A digital microfluidic system for loop-mediated isothermal amplification and sequence specific pathogen detection.

A digital microfluidic (DMF) system has been developed for loop-mediated isothermal amplification (LAMP)-based pathogen nucleic acid detection using specific low melting temperature (Tm) Molecular Beacon DNA probes. A positive-temperature-coefficient heater with a temperature sensor for real-time thermal regulation was integrated into the control unit, which generated actuation signals for droplet manipulation. To enhance the specificity of the LAMP reaction, low-Tm Molecular Beacon probes were designed within the single-stranded loop structures on the LAMP reaction products. In the experiments, only 1 µL of LAMP reaction samples containing purified Trypanosoma brucei DNA were required, which represented over a 10x reduction of reagent consumption when comparing with the conventional off-chip LAMP. On-chip LAMP for unknown sample detection could be accomplished in 40 min with a detection limit of 10 copies/reaction. Also, we accomplished an on-chip melting curve analysis of the Molecular Beacon probe from 30 to 75 °C within 5 min, which was 3x faster than using a commercial qPCR machine. Discrimination of non-specific amplification and lower risk of aerosol contamination for on-chip LAMP also highlight the potential utilization of this system in clinical applications. The entire platform is open for further integration with sample preparation and fluorescence detection towards a total-micro-analysis system.

Drug screening of cancer cell lines and human primary tumors using droplet microfluidics.

Precision Medicine in Oncology requires tailoring of therapeutic strategies to individual cancer patients. Due to the limited quantity of tumor samples, this proves to be difficult, especially for early stage cancer patients whose tumors are small. In this study, we exploited a 2.4 × 2.4 centimeters polydimethylsiloxane (PDMS) based microfluidic chip which employed droplet microfluidics to conduct drug screens against suspended and adherent cancer cell lines, as well as cells dissociated from primary tumor of human patients. Single cells were dispersed in aqueous droplets and imaged within 24 hours of drug treatment to assess cell viability by ethidium homodimer 1 staining. Our results showed that 5 conditions could be screened for every 80,000 cells in one channel on our chip under current circumstances. Additionally, screening conditions have been adapted to both suspended and adherent cancer cells, giving versatility to potentially all types of cancers. Hence, this study provides a powerful tool for rapid, low-input drug screening of primary cancers within 24 hours after tumor resection from cancer patients. This paves the way for further technological advancement to cutting down sample size and increasing drug screening throughput in advent to personalized cancer therapy.

Ammonium tetrathiomolybdate treatment targets the copper transporter ATP7A and enhances sensitivity of breast cancer to cisplatin.

Cisplatin is an effective breast cancer drug but resistance often develops over prolonged chemotherapy. Therefore, we performed a candidate approach RNAi screen in combination with cisplatin treatment to identify molecular pathways conferring survival advantages. The screen identified ATP7A as a therapeutic target. ATP7A is a copper ATPase transporter responsible for intercellular movement and sequestering of cisplatin. Pharmaceutical replacement for ATP7A by ammonium tetrathiomolybdate (TM) enhanced cisplatin treatment in breast cancer cells. Allograft and xenograft models in athymic nude mice treated with cisplatin/TM exhibited retarded tumor growth, reduced accumulation of cancer stem cells and decreased cell proliferation as compared to mono-treatment with cisplatin or TM. Cisplatin/TM treatment of cisplatin-resistant tumors reduced ATP7A protein levels, attenuated cisplatin sequestering by ATP7A, increased nuclear availability of cisplatin, and subsequently enhanced DNA damage and apoptosis. Microarray analysis of gene ontology pathways that responded uniquely to cisplatin/TM double treatment depicted changes in cell cycle regulation, specifically in the G1/S transition. These findings offer the potential to combat platinum-resistant tumors and sensitize patients to conventional breast cancer treatment by identifying and targeting the resistant tumors' unique molecular adaptations.

CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth.

Polarisome is a protein complex that plays an important role in polarized growth in fungi by assembling actin cables towards the site of cell growth. For proper morphogenesis, the polarisome must localize to the right place at the right time. However, the mechanisms that control polarisome localization remain poorly understood. In this study, using the polymorphic fungus Candida albicans as a model, we have discovered that the cyclin-dependent kinase (CDK) Cdc28 phosphorylates the polarisome scaffold protein Spa2 to govern polarisome localization during both yeast and hyphal growth. In a yeast cell cycle, Cdc28-Clb2 phosphorylates Spa2 and controls the timing of polarisome translocation from the bud tip to the bud neck. And during hyphal development, Cdc28-Clb2 and the hyphal-specific Cdc28-Hgc1 cooperate to enhance Spa2 phosphorylation to maintain the polarisome at the hyphal tip. Blocking the CDK phosphorylation causes premature tip-to-neck translocation of Spa2 during yeast growth and inappropriate septal localization of Spa2 in hyphae and abnormal hyphal morphology under certain inducing conditions. Together, our results generate new insights into the mechanisms by which fungi regulate polarisome localization in the control of polarized growth.

Regulation of Rfa2 phosphorylation in response to genotoxic stress in Candida albicans.

Successful pathogens must be able to swiftly respond to and repair DNA damages inflicted by the host defence. The replication protein A (RPA) complex plays multiple roles in DNA damage response and is regulated by phosphorylation. However, the regulators of RPA phosphorylation remain unclear. Here, we investigated Rfa2 phosphorylation in the pathogenic fungus Candida albicans. Rfa2, a RFA subunit, is phosphorylated when DNA replication is inhibited by hydroxyurea and dephosphorylated during the recovery. By screening a phosphatase mutant library, we found that Pph3 associates with different regulatory subunits to differentially control Rfa2 dephosphorylation in stressed and unstressed cells. Site-directed mutagenesis revealed T11, S18, S29, and S30 being critical for Rfa2 phosphorylation in response to genotoxic insult. We obtained evidence that the genome integrity checkpoint kinase Mec1 and the cyclin-dependent kinase Clb2-Cdc28 mediate Rfa2 phosphorylation. Although cells expressing either a phosphomimetic or a non-phosphorylatable version of Rfa2 had defects, the latter exhibited greater sensitivity to genotoxic challenge, failure to repair DNA damages and to deactivate Rad53-mediated checkpoint pathways in a dosage-dependent manner. These mutants were also less virulent in mice. Our results provide important new insights into the regulatory mechanism and biological significance of Rfa2 phosphorylation in C. albicans.